GUS Transformants Research Articles

RNA In Situ Hybridization of Paraffin Sections to Characterize the Multicellular Compartmentation of Plant Secondary Metabolisms.

As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus. RNA in situ hybridization has proven a key technological approach to unravel this complex spatial organization. Pioneer work in 1999 discovered the involvement of epidermis and laticifer/idioblasts in the intermediate and late steps of the pathway, respectively. The localization of the early steps of the pathway to the internal phloem-associated parenchyma later came to complete the three-tissular block organization of the pathway. Since then, RNA in situ hybridization was routinely used to map the gene expression profile of most of the nearly 30 genes involved in this pathway. We introduce here a comparison of advantages and drawbacks of in situ hybridization and more popular promoter: GUS strategies. Two main advantages of in situ hybridization are the suitability to any plant species and the direct localization of transcripts rather than the localization of a promoter activity. We provide a step-by-step protocol describing every details allowing to reach a medium throughput including riboprobe synthesis, paraffin-embedded plant tissue array preparation, prehybridization, in situ hybridization, stringent washing and immunodetection of hybridized probes, and imaging steps. This should be helpful for new comers willing to domesticate the technique. This protocol has no species limitation and is particularly adapted to the increasingly studied model, nonmodel species, nonamenable to promoter::GUS transformation, such as C. roseus.

Functional characterization of three MATE genes in relation to aluminum-induced citrate efflux from soybean root

Citrate transporters responsible for Aluminum (Al)-induced citrate efflux have not been identified in soybean. Three soybean multi-drug and toxic compound extrusion (MATE) family genes were characterized by transcriptional expression, subcellular localization and overexpression experiments. GmMATE75, GmMATE79 and GmMATE87 are localized to plasma membrane. Their over-expression respectively resulted in more citrate efflux and less Al contents in soybean hairy roots, alleviated root elongation inhibition in Arabidopsis and partially restored root growth in atmate mutant under Al stress. Al increased their transcriptional expression at either the root apex or the base zone. Cu2+, Cd2+, La3+ increased the expression of GmMATE79 and GmMATE87. Ten day of –Fe culture increased the expression of GmMATE75 and GmMATE79. Al treatment extended β-glucuronidase (GUS) staining from central cylinder to cortical and epidermis cells for pGmMATE75::GUS or pGmMATE79::GUS soybean hairy roots transformation. But GUS staining restricted within central cylinder for pGmMATE87::GUS transformation with or without Al treatment. GmMATE75, GmMATE79 and GmMATE87 are plasma-membrane-localized citrate transporters and have capabilities to increase citrate efflux. They played different role in Al-induced citrate secretion from soybean because their different tissue localization and expression patterns.

Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn

Microprojectile bombardment mediated genetic transformation parameters have been standardized for seed derived callus of Eleusine coracana. Plasmid pCAMBIA 1381 harboring hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (gus A) as reporter gene, was used for the optimization of gene transfer conditions. The transient GUS expression and survival of putative transformants were taken into consideration for the assessment of parameters. Optimum conditions for the microprojectile bombardment mediated genetic transformation of finger millet were 1,100 psi rupture disk pressure with 3 cm distance from rupture disk to macrocarrier and 12 cm microprojectile travel distance. Double bombardment with gold particles of 1.0 μm size provided maximum transient GUS expression and transformation efficiency. Osmotic treatment of callus with 0.4 M sorbitol enhanced efficiency of particle bombardment mediated genetic transformation. Regenerative calli were bombarded at optimum conditions of bombardment and placed on regeneration medium with hygromycin to obtain transformed plants. The integration of hptII and gus A genes was confirmed with PCR amplification of 684 and 634 bp sizes of the bands respectively from putative transformants and Southern blot hybridization using PCR amplified DIG labeled hptII gene as probe. PCR analysis with hptII gene specific primers indicated the presence of transgene in T1 generation plants. Thus a successful genetic transformation system was developed using particle bombardment in E. coracana with 45.3% transformation efficiency. The protocol will be helpful for the introgression of desired genes into E. coracana.

Regulation of root growth by auxin and ethylene is influenced by phosphate supply in white clover (Trifolium repens L.)

The influence of P-supply on root system architecture (primary root length, number and total length of lateral roots) through the effects of ethylene (ACC) and auxin [1-naphthylacetic acid (NAA)] has been examined in the legume white clover (Trifolium repens L.). Higher concentrations (1 and 10 μM) of ACC and NAA (100 nM) inhibited growth, while lower concentrations (100 nM ACC, 5 nM NAA) either had no effect or stimulated growth in P-sufficient (1 mM Pi) roots. In response to low (10 μM) P, a stimulation of primary root growth, number of lateral roots and mean length of lateral roots was observed, while a super-stimulation of these growth parameters occurred in response to subsequent 100 nM ACC treatment suggesting that the low P treatment increased the sensitivity of the roots to ethylene. Examination of the primary roots of DR5p::GUS transformants suggests that this change in sensitivity induced by low P occurs through the promotion of auxin signalling/transport to the root apex. These results are discussed in terms of the role of ethylene and the significance of changes in sensitivity to the hormone in modulating root system architecture in response to low P-supply.

High-throughput screening for plant defense activators using a .BETA.-glucuronidase-reporter gene assay in Arabidopsis thaliana

To develop a screening system for plant defense activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we utilized a GUS histochemical staining assay using promoters of the defense-related genes, PR-1 and PDF1.2. We can perform about 1,000 screenings per week per person by this high- throughput screening method. This GUS assay for plant defense activator candidates was evaluated by QRT-PCR analysis to elucidate the functions of the plant defense activators in detail. In the present preliminary screening, we evaluated two hundred chemicals chosen at random. Some chemicals induced GUS activity in a PR-1 promoter::GUS transformant, i.e., abietic acid, allose, glycine, and thymol. The induction of PR-1 expression by the treatments with these chemicals was confirmed using QRT-PCR. The foliar treatment with abietic acid 1 d prior to inoculation with the fungal pathogen Colletotrichum higginsianum led to a significant reduction of necrotic surface area compared with distilled water treated controls, as observed 6 d after inoculation. These results suggest that this GUS histochemical staining assay is an effective and available screening system for plant defense activators.

Optimization of Parameters for GUS Gene Transformation of Porphyra yezoensis by Particle Bombardment

We optimized the biological and physical parameters for DNA delivery into thalli of the red alga Porphyra yezoensis using a particle bombardment device. The efficiency of transformation was determined using the (GUS) assay. The optimal helium pressure, distance of tungsten particle flight, and ratio of DNA to tungsten particles were , 8 cm, and tungsten, respectively. During bombardment, osmotic treatment with a mixture of 0.6 M mannitol and sorbitol increased the efficiency of GUS transformation. After 2 days, the blue color indicating GUS activity was observed using a histochemical assay.

Comparison of genetic transformation in Morus alba L. via different regeneration systems

Three different regeneration systems, viz. direct regeneration of adventitious shoot buds from explant, regeneration through callus cultures and somatic embryos were compared to see their effect on transfer of neomycin phosphotransferase (nptII) and beta-glucuronidase (GUS) reporter gene (gus) to Morus alba clone M5, through Agrobacterium tumefaciens mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency. The highest transformation frequency of 18.6% was obtained using direct induction of adventitious shoot buds. Expression and presence of transgene were assayed histochemically and through polymerase chain reaction. Southern analysis of GUS and PCR positive transformants confirmed stable integration of transgenes with two to four copy numbers. The selected transformants showed normal phenotype under in vitro and field conditions.

Proteomics to identify resistance factors in corn-a review

The host resistance strategy for eliminating aflatoxins from corn has been advanced by the discovery of natural resistance traits such as proteins. This progress was aided by the development of a rapid laboratory-based kernel screening assay (KSA) used to separate resistant from susceptible seed, and for investigating kernel resistance.A. flavus GUS transformants have also been used, in conjunction with the KSA, to assess the amount of fungal growth in kernels and compare it with aflatoxin accumulation. Several proteins associated with resistance (RAPs) have been identified using 1 D PAGE. However, proteomics is now being used to further the discovery of RAPs. This methodology has led to the identification of stress-related RAPs as well as other antifungals. Characterization studies being conducted, including RNAi gene silencing experiments, may confirm roles for RAPs in host resistance.

Expression of 1-Aminocyclopropane-1-Carboxylate (ACC) Oxidase Genes During the Development of Vegetative Tissues in White Clover (Trifolium repens L.) is Regulated by Ontological Cues

Four 5' flanking sequences, comprising the 5'-UTR and upstream promoter region, have been isolated and cloned from the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family of white clover (Trifolium repens L.), and designated TR-ACO1p, TR-ACO2p, TR-ACO3p and TR-ACO4p. Southern analysis confirmed that these sequences correspond to four distinct genes. The four corresponding genomic sequences have also been isolated and each shown to be comprised of four exons interspersed by three introns. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into white clover. Here, the TR-ACO1 5' flanking sequence directs highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the TR-ACO2 5' flanking sequence directs expression in both younger, mature green and in ontologically ageing tissue. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. The TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, but all four 5' flanking sequences directed expression in the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule, with the primacy of each promoter determined by the ontological age of the tissues. These data suggest that in vegetative tissue development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.

Root-synthesized cytokinin in Arabidopsis is distributed in the shoot by the transpiration stream

To clarify how root-synthesized cytokinins (CKs) are transported to young shoot organs, CK distribution patterns were analysed in free-CK-responsive ARR5::GUS transformants of Arabidopsis thaliana (L.) Heynh. together with free plus bound CKs using specific CK monoclonal antibodies. Plants were subjected to two different growth conditions, completely protected from any air movement, or exposed to gentle wind 3 h before harvesting. In wind-protected plants the strongest ARR5::GUS expression was found in the root cap statocytes, spreading upwards in the vascular cylinder. This pattern in roots was congruent with that found by CK immunolocalization. Shoots of wind-protected plants displayed either no or only low ARR5::GUS expression in the stem vascular bundles, nodal ramifications, and the bases of flower buds; shoot vascular bundles showed patterns of acropetally decreasing staining and the apical parts of buds and leaves were free from ARR5::GUS expression. In wind-exposed plants ARR5::GUS expression was considerably increased in shoots, also in basal-to-apical decreasing gradients. Immunolabelled shoots showed differential staining, with the strongest label in the vascular bundles of stems, leaves, and buds. The fact of the apparent absence of free CK in the buds of wind-protected plants and the typical upward decreasing gradients of free and conjugated CKs suggest that the bulk of the CK is synthesized in the root cap, exported through the xylem and accumulates at sites of highest transpiration where cuticles do not yet exist or do not protect against water loss.

In Arabidopsis thaliana, the invertase inhibitors AtC/VIF1 and 2 exhibit distinct target enzyme specificities and expression profiles

Plant cell wall (CWI) and vacuolar invertases (VI) play important roles in carbohydrate metabolism, stress responses and sugar signaling. Addressing the regulation of invertase activities by inhibitor proteins (C/VIF, c ell wall/ v acuolar i nhibitor of f ructosidase), we have identified two C/VIFs from Arabidopsis thaliana. AtC/VIF1 showed specific inhibition of VI activity, whereas AtC/VIF2 inhibited both, CWI and VI. Expression analysis revealed that expression of AtC/VIF1 was restricted to specific organs, AtC/VIF2, however, was weakly expressed throughout plant development. Promoter::GUS transformants confirmed pronounced differences of tissue/cell type-specific expression between both isoforms. Growth of an AtC/VIF1 T-DNA KO mutant was unaffected, but VI activity and hexose content were slightly increased.

A poly(U) motif in the 5′ untranslated region enhances the translational efficiency of β-glucuronidase mRNA in transgenic tobacco

We examined how the base composition of the 5′ untranslated region (UTR) modifies the translational efficiency of plant mRNAs with the aid of chimeric reporter constructs, and transient and stable transformation of tobacco. We inserted 20 base homopolymers of G, A, T and C into the 5′ UTR of a β-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S promoter, and the resultant constructs were introduced into tobacco seedlings by particle bombardment. The transient GUS activity of the seedlings became higher only when the (T) 20 motif was inserted. To elucidate how the (T) 20 motif altered gene expression, we introduced the (T) 20-inserted GUS construct into tobacco plants by Agrobacterium-mediated stable transformation and examined its expression in comparison with the GUS construct with no insert. The apparent GUS activity of the (T) 20-GUS transgenic plants was 4.5-fold higher than that of the control GUS transformants, although the GUS mRNA levels were similar between these transgenic lines. The GUS activity/GUS mRNA ratio was 5.6-fold higher in the (T) 20-inserted GUS transformants than in the controls. This indicates that the (U) 20 motif inserted into the 5′ UTR enhances the translational efficiency of the GUS reporter mRNA in tobacco cells.

Elevation of transgene expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the interactions of six promoters with the RB7 3' MAR.

We have analyzed effects of a matrix attachment region (MAR) from the tobacco RB7 gene on transgene expression from six different promoters in stably transformed tobacco cell cultures. The presence of MARs flanking the transgene increased expression of constructs based on the constitutive CaMV 35S, NOS, and OCS promoters. Expression from an induced heat shock promoter was also increased and MARs did not cause expression in the absence of heat shock. There was also no effect of MARs on the pea ferredoxin promoter, which is not normally expressed in this cell line. Importantly, most transgenes flanked by RB7 MAR elements showed a large reduction in the number of low expressing GUS transformants relative to control constructs without MARs.

GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.

Increasing Transient and Subsequent Stable Transgene Expression in Chrysanthemum Following Optimization of Particle Bombardment and Agroinfection Parameters.

Transient and stable transgene expression was maximized in in vitro and greenhouse-derived chrysanthemum ‘Lineker’ and ‘Shuhou-no-chikara’ stem explants. In Agroinfection, Agrobacterium tumefaciens LBA4404-with either pBI121 or pKT2-or AGL0 (with pKT3) were utilized, while in particle bombardment pSKGN1 was utilized. Transformation efficiency was affected by both the gene introduction method and its experimental parameters and the origin and developmental state of the explant. There was a decrease in GUS focal points with a simultaneous increase in the number of blue staining areas for both ‘Lineker’ and ‘Shuhou-no-chikara’ in both Agroinfection (pKT2 and pKT3) and particle bombardment (pSKGN1) over time (0, 24, 48, 72 h and 1, 2 and 4 weeks). GUS transformation efficiency was 6.6, 26.89, 25.0 and 11.84% for Agroinfection, bombardment, sonication and Agrolistics, respectively for ‘Lineker’, and 0, 2.17, 0 and 7.69%, respectively for ‘Shuhou-no-chikara’, the highest level of stable GUS transgene expression occurring in thevenation.

The use of a GUS transformant of Trichoderma harzianum, strain T3a, to study metabolic activity in the spermosphere and rhizosphere related to biocontrol of Pythium damping-off and root rot

The activity of Trichoderma harzianum in the spermosphere and rhizosphere of different plant species was studied by use of a β-glucuronidase (GUS) transformant (strain T3a). Hereby, direct observation of micro-habitats supporting metabolic activity of T. harzianum is reported. Germination of conidia and mycelial growth were not supported by exudates from healthy roots of various ages. Instead, growth and activity of T. harzianum depended on access to dead organic substrates such as seed coats, decaying roots, and wounds, including those caused by infecting pathogens. A correlation between the GUS activity of T. harzianum and the biomass of Pythium ultimum in infected roots was established. On the basis of our observations, we suggest that the biocontrol ability of T. harzianum involves competition with the pathogen for substrates including the seed coat, and wounded or infected root tissue.

Analysis of gene promoters for two tomato polygalacturonases expressed in abscission zones and the stigma.

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes;Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to beta-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5'-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.

Antifungal Activity Toward Fusarium culmorum in Soluble Wheat Extracts

ABSTRACT This study investigated antifungal activity in soluble extracts from seed of a range of wheat cultivars differing in susceptibility to Fusarium head blight. Antifungal activity was assessed in terms of beta-D-glucuronidase (GUS) activity of a Fusarium culmorum GUS transformant using a sensitive laboratory assay. Significant antifungal activity was detected in seed extracts from WEK0609, CM 820036, and Arina. Initial characterization of the Arina seed extract indicated that it contained antifungal proteinaceous compounds. The Arina extract yielded two (60 and 80%) ammonium sulfate fractions containing inhibitory compounds. Gel filtration chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of antifungal fractions showed that the antifungal activities detected in the Arina 60 and 80% ammonium sulfate fractions were associated with putative proteinaceous compounds with apparent molecular masses of approximately 60 and 28 kDa, respectively.

Factors affecting soybean cotyledonary node transformation.

Cotyledonary node transformation efficiency was evaluated using a sonication assisted Agrobacterium-mediated transformation (SAAT) protocol, three dissimilar A. tumefaciens strains, and explants derived from 28 diverse cultivars and/or genotypes of soybean [Glycine max (L.) Merr.]. The explants were evaluated at 10 and 45 days after co-cultivation for transformation with a binary vector containing both a GUS-intron gene and an NPTII selectable marker. The best overall strain of A. tumefaciens was determined to be KYRT1 based on stable GUS transformation of soybean cotyledonary node explants measured at the terminal 45 day evaluation point. SAAT did not increase stable transformation at 45 days post co-cultivation. SAAT was determined to significantly decrease shoot proliferation of some genotypes, but it is unclear what effect this may have on the recovery of transformed shoots. Significant differences were also detected between genotypes for transformation and shoot proliferation frequency.

A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment

The binding or precipitation of DNA onto gold or tungsten microcarriers represents one of the most crucial steps for gene transfer via the particle bombardment process. We have developed a simple and rapid method to monitor DNA precipitation onto microcarriers before delivery to intact cells or tissues. Binding of DNA constructs to different microcarriers was evaluated with relative fluorescence values using a dedicated fluorometer. Significantly greater precipitation was detected using gold vs. tungsten microcarriers. Addition of glycerol resulted in a 46% increase in precipitation. A 42% difference in precipitation was observed using two different brands of polyproplyene tubes. Fluorescence values dropped 10-50% 3 hr after initial precipitation. Fluorescence values were correlated with the number of transient GUS transformants of rice (Oryza sativa, L.) cells. Precipitation with PEG gave higher fluorescent values and GUS transformants than a similar method without PEG. Results from these experiments indicate that fluorescence measurements are an effective and rapid method to monitor DNA precipitation for particle bombardment experiments.